The present invention relates generally to methods for determining the presence of active periodontal disease in mammals through assaying gingival crevicular fluid for the presence of intracellular enzymes. Specifically, the invention relates to methods for the determination of periodontal disease through assaying crevicular fluid for the presence of elevated levels of the enzyme L-alanine aminotransferase (E.C. 2.6.1.2) (ALT).
ALT is an intracellular enzyme widely distributed among mammalian tissues. Following acute tissue injury in the course of disease, trauma or toxicity, damaged cells release ALT into the circulation, interstitial fluid, inflammatory exudate and other bodily fluids. Elevated ALT in humans is indicative of tissue injury, most often associated with diseases of the liver.
Periodontal diseases are inflammatory diseases of microbial etiology affecting the supporting tissues of the teeth. The term "periodontal disease" encompasses two major and distinct subclasses of disease, gingivitis and periodontitis. Gingivitis is characterized by inflammation of the gums without bone loss or loss of connective tissue attachment. Gingivitis is a precondition for, but does not necessarily lead to, periodontitis which is characterized by progressive formation of periodontal pockets between the gum tissue and tooth, resulting from loss of connective tissue attachment and bone loss, eventually leading to tooth loss. Presently available methods of measuring periodontal disease include subjective observational indices such as those of Loe, H. and P. Silness, Acta Odont. Scand., 21:533 (1963) for gingivitis, and Ramfjord, S., J. Periodontol., 30:602 (1967) for periodontitis. These indices for periodontitis are based on criteria such as bleeding on gentle probing, pocket depth, attachment loss, and radiographic evidence of bone loss. Unfortunately, these clinical indicators, with the exception of bleeding on probing, are generally acknowledged to be reflective of past disease and prior damage. Of these indicators, only bleeding on probing (bleeding of gum tissue due to probing of the gum line or pocket with a hard instrument, e.g., probe or curet) has been claimed to correlate with active periodontal disease. Nevertheless, bleeding itself is a subjective indicator of disease and the diagnostic value of bleeding on probing has been questioned, as such bleeding appears to be associated with a high proportion of false positive indications of periodontal disease. See Haffajee, A. D., S. S. Socransky and J. M. Goodson, J. Clin. Perio., 10:257-265 (1983).
Other methods have been proposed for the diagnosis of periodontal disease. Because both gingivitis and periodontitis are characterized by accumulation and flow of crevicular fluid (a transudate of serum) at the gingival sulcus and pockets, measurement of the volume of crevicular fluid present at a site has been proposed as a diagnostic method for the detection of periodontal disease. An instrument known as the Periotron (Harco Electronics Ltd.; Winnipeg, Canada) makes use of this principle by galvanometrically measuring the volume of crevicular fluid absorbed by small strips of porous material known as Periopaper (Harco; Tustin, California) which are inserted into the crevicular space between the tooth and gum.
Still other methods relate to analysis of components of crevicular fluid for the diagnosis of periodontal disease. Kornman, J. Period. Res., 22, (1987) discloses methods correlating the presence of collagenase in crevicular fluid with the severity of periodontal disease. A device (Periocheck, Advanced Clinical Technologies, Inc., Westwood, Mass.) is available which assays for neutral proteases to determine the presence of periodontal disease. The source of both collagenase and neutral proteases has been suggested to be polymorphonuclear leucocytes migrating into the crevice. Other components of crevicular fluid such as chondroitin-4-sulfate, the presence of which is considered indicative of bone destruction, have been found to differ in crevicular fluid associated with gingivitis and crevicular fluid associated with periodontitis. Prostaglandin E.sub.2, a mediator of inflammation, has also been indicated to be associated more closely with periodontitis than with gingivitis.
The enzyme aspartate aminotransferase (AST) is an intracellular enzyme widely distributed among tissues and organs of the body. Elevated levels of AST in the blood and other bodily fluids is indicative of tissue inflammation and cellular damage. In particular, AST has been used in the detection of disorders of the liver, heart and skeletal muscles. The ratio of AST to ALT (formerly SGOT/SGPT ratio) is useful in assessing the extent of hepatic damage; the greater the ratio, the worse the damage.
It has been found that elevated levels of AST in crevicular fluid is highly correlative with the presence of active periodontal disease. (See abstracts presented at the American Association for Dental Research Meetings, Cincinnati, Ohio, March 17-20, 1983: Crawford, J. M. , S. Mukherjee, D. A. Chambers and R. Cohen, Abstract No. 241; and Mukherjee, S., J. Crawford, D. A. Chambers and R. Cohen, Abstract No. 242; Chambers, D. A., J. M. Crawford, S. Mukherjee and R. Cohen, J. Periodon., 55, No. 9, 526-530, Sept. 1984). The Crawford, et al. abstract discloses a study with dogs in which gingivitis and periodontitis were induced experimentally. Specifically, gingival health was established in five beagles and gingivitis was then allowed to develop for four weeks by introduction of a soft diet and withdrawal of brushing. Periodontitis was then induced by ligation of the dogs' teeth. Crevicular fluid samples were collected at weekly intervals in volumetric capillary tubes after isolation and drying of teeth. The abstract indicates that crevicular fluid obtained during the incidence of experimental periodontitis contained-d concentrations of AST (3209.+-.1435 SFU/ml) approximately ten-fold higher at their peak than prior to ligation (468.+-.164 SFU/ml) and further that crevicular fluid during the incidence of experimental gingivitis contained approximately ten-fold higher concentrations of AST than in serum (41.+-.4 SFU/ml).
The Chambers, et al., J. Periodont. publication describes the dog study in greater detail and notes that the: average AST levels in crevicular fluid correlated neither with clinical estimations of attachment levels nor with gingival inflammation. The article did note, however, that the peak of AST activity occurring 2 weeks after ligation of teeth did coincide with the period of high levels of soft tissue destruction and osteoclast activity reported in the beagle dog model and with the period of active bone resorption in ligature-induced periodontitis in the monkey model. The article also disclosed that the level of AST in crevicular fluid did not correlate with enzyme levels in dental plaque, suggesting the absence of bacterial origin for the enzyme.
The Mukherjee Abstract describes measurement of AST levels in human crevicular fluid collected in volumetric capillary tubes from areas diagnosed as having gingivitis or periodontitis according to the periodontal disease index (PDI) of Ramfjord. Disease activity indicated by the presence or absence of bleeding upon probing was also noted. AST concentrations of crevicular fluid collected from areas showing no bleeding on probing =0 SFU/ml (N=4), minimal bleeding =464.+-.113 SFU/ml (N=4) and definite bleeding 595.+-.192 SFU/ml (N=6). The analysis of data sorted according to gingivitis and periodontitis showed 363.+-.182 SFU/ml (N=4) and 424.+-.119 SFU/ml (N=3), respectively. The Abstract notes that the level of AST in crevicular fluid may correlate with the disease activity as determined by bleeding on probing.
While the references fail to demonstrate a specific positive association between elevated AST activity in gingival crevicular fluid and either attachment loss or gingival inflammation, they do indicate that there exists a general association between elevated AST levels in gingival crevicular fluid and periodontal disease activity as determined by bleeding on probing. Chambers, European patent application No. 151,536 published Aug. 14, 1985 based on U.S. patent application Ser. No. 575,552 filed Jan. 31, 1984, the disclosure of which is hereby incorporated by reference, relates to the work embodied in the article and abstracts and the recognition of the general relationship between elevated AST levels and periodontal disease activity. The application describes diagnostic methods based on the recognition that the presence of elevated levels of AST in crevicular fluid is predictive of a high probability of progressive, as opposed to non-progressive, periodontal disease and corresponding tissue damage.
According to a method described in the Chambers patent application, the crevicular fluid is collected from the gingival sulcus by means such as a microsyringe, capillary tube or absorbent strip. The volume of material is measured and the concentration of AST in the collected sample of crevicular fluid is determined by either colorimetric or immunological assay. The patent application describes a method for determining the presence of active periodontal disease in mammals comprising assaying crevicular fluid for the presence of elevated levels of aspartate aminotransferase. The application defines elevated levels as being an amount of AST substantially in excess of the level of AST normally found in the blood stream of healthy adults of the species being tested which ranges from about 4 to about 32 milli-International Units/ml (mIU/ml) depending upon the precise testing protocol used.
In addition to the work by the Chambers, et al. group on AST, the relationship between other tissue and bacterial enzymes and periodontal diseases has been studied. Lamster, et al., J. Periodontal., 56, 139-147 (1985), disclose studies assessing crevicular fluid volume and activity of the enzymes lactate dehydrogenase (LDH), .beta.-glucuronidase (BG) and arylsulfatase (AS) in crevicular fluid during the development of experimental gingivitis. Bang, et al., Helv. Odont. Acta., 16:89 (1972); Weinstein, et al., Archs. Oral Biol., 17:375 (1972) and Snyder, et al., J. Dent. Res., 62:196 (1983) relate to the presence of LDH in gingival crevicular fluid and the correlation of LDH with parameters of periodontal disease. Bang, et al., Archs. Oral Biol., 15:445-451 (1970) relate to the correlation of BG with gingival inflammation.
LDH is said to be derived primarily from cells in the sulcular epithelium, but fibroblasts and polymorphonuclear leucocytes that lyse in the crevice also contribute to the LDH pool. BG is said to be primarily derived from degradation of lysosomal granules of infiltrating polymorphonuclear leucocytes and macrophages. The pattern of AS activity was characterized as being between that of LDH and BG with sources of this enzyme including polymorphonuclear leucocytes, mast cells and fibroblasts.
Crevicular fluid "resting" volumes were determined by inserting a filter paper strip into the gingival sulcus until mild resistance was felt and leaving the strip in place for 30 seconds before removing the strip and determining the volume of fluid absorbed. After removal of the strip, the crevicular fluid "flow" volume was determined by waiting 30 seconds and inserting a second filter paper strip into the site for 3 seconds. Analysis of data collected from subjects subjected to experimental gingivitis indicated that while clinical inflammation increased during the 4 weeks of the study, the concentrations and total activity (concentration times sample volume) of BG and AS rose during the onset of gingivitis but peaked or leveled off after reaching a maximum 2 or 3 weeks into the study. The data indicated that an increase in fluid volume without a corresponding increase in BG or AS activity occurred during the latter part of the trial. The increase in LDH concentration and total activity during the experiment was not dramatic and was consistent with an earlier experiment wherein the concentration of LDH in crevicular fluid was higher in subjects with healthy gingiva than with mildly inflamed gingiva Lamster, et al. also suggested that reporting of crevicular fluid constituent data in terms of concentration alone might be inadequate and that it might be desirable to report enzyme data in terms of both concentration and total activity of the sample.
Lamster, et al., J. Clin. Periodontal., 13, 799-804 (1986), present data wherein LDH, BG and AS concentrations and total activities for 30 second samples were assayed for a group of periodontitis patients and a control group. Negative or low positive correlation coefficients were determined between enzyme concentrations and gingival index (GI) and probing depth. On the other hand, a "modest, but not absolute" correlation between increasing severity of pathology and total enzyme activity for a 30 second sample was suggested by the data. Lamster, et al. suggested, therefore, that total activity in a standardized sample might be a more appropriate means of reporting crevicular fluid constituent data.
More recently, Chambers, et al. U.S. patent application Ser. No. 262,995 filed Oct. 26, 1988, the disclosure of which is incorporated herein by reference, revealed the results of an experimental study of ligature-induced periodontitis in dogs and a longitudinal study of periodontitis patients, regarding measurement of activity of AST in gingival crevicular fluid (GCF). In the application, it was shown that the total activity of AST present in a GCF sample taken for a selected brief period of time provided a better correlation with periodontal disease activity than did assaying for the AST concentration of GCF. In addition, it was found that the total AST activity in the GCF samples was indicative of both the severity and type of periodontal disease, either gingivitis or periodontitis.
In spite of the various advances made in the art, a simple, reliable means for an enzymatic determination of the presence of periodontal disease is desired. Such a method might be used for diagnosis of such disease or for determination of the efficacy of treatment of that periodontal disease condition. The monitoring of periodontal disease is a nontrivial concern because of the serious nature of continuing or repeating treatments for periodontitis, which involve administration of drugs, root planing or surgery. Current methods such as monitoring of the clinical parameters of probing depths and radiographic interpretations of the bone provide only a belated evaluation of treatment efficacy. Accordingly, improved approaches are clearly desired.